The tumor vasculature differs from that of normal tissues with respect to organization, structure and function. Tumors induce new vessels by secreting vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and other angiogenic cytokines. Angiogenesis follows a consistent series of steps, two of which are increased microvascular permeability and generation of enlarged, thin-walled, pericyte-poor "mother" vessels which subsequently spawn smaller caliber "daughter" vessels. Recently, VPF/VEGF, introduced into normal adult tissues with an adenoviral vector, was found to induce both of these steps. The present application addresses a number of critical questions: How does VPF/VEGF, whether tumor-secreted or delivered by an adenoviral vector, elicit mother and daughter vessels? How does tumor vessel generation differ from normal vascular development? Can a more normal vasculature be induced in tissues by appropriate combinations of cytokines? Can tumors be induced to form a more normal vascular supply? If so, what would the consequences be for tumor behavior? What is the composition of the vesiculo-vacuolar organelle (VVO), the newly described endothelial cell organelle implicated in extravasation of plasma and plasma proteins from tumor vessels? How do VVOs relate to caveolae? How do VV0s open so as to allow the passage of circulating macromolecules? Can permeability to macromolecules of the type induced by mediators such as VPF/VEGF in vivo be replicated in cultured EC? We will address these questions with the following four specific aims: 1. Elucidate the steps and mechanisms by which tumors induce "mother" and "daughter" vessels. 2. Identify cytokine combinations that induce a more normally differentiated vasculature than that induced by VPF/VEGF alone. Determine the consequences of vessel differentiation for tumor growth. 3. Determine the composition of and purify VVOs. 4. Develop an in vitro assay that is responsive to VPF/VEGF and other vasoactive mediators and that measures the permeability of cultured EC to macromolecules in a manner that mimics the in vivo response with respect to mechanism, sensitivity and kinetics.